The concern over the possible reemergence of severe invasive diseases caused by Streptococcus pyogenes (group A streptococcus) has focused attention on the role of its secreted cysteine protease in pathogenesis. Unfortunately and despite extensive application of modern genetic technology, various studies have both supported and refuted an important role for the protease in promoting disease. Thus, the role of the cysteine protease in pathogenesis remains unclear. It is likely that much of the controversy has arisen because the biogenesis of the cysteine protease is only poorly understood. However, it is clear that this process is highly regulated, both at the level of transcription and at the level of processing the secreted inactive pro-protein to the active form. My lab has begun to address this issue and developed a novel transposon to identify three loci required for expression of proteolytic activity. Designated as Rop loci (regulation of protease), ropB is an activator of transcription of the gene which encodes the protease and is a member of the emerging Rgg-like family of regulators of which very little is currently known. In contrast, ropA contributes to secretion and processing of the protease and encodes a homologue of Trigger Factor, a peptidyl-prolyl isomerase and putative chaperone, which is highly conserved in most bacterial species, but of unknown function. Preliminary studies have shown that RopA acts both to assist in targeting the protease to the secretory pathway and in promoting the ability of the proprotein to establish an active conformation upon secretion. The Specific Aims will address 1.) the function of ropC, including whether it is involved in transcription, secretion or processing of protease; 2.) Further characterization of RopB, focusing on its possible interactions with the multiple promoters for the gene which encodes the protease; 3.) an in-depth analysis of the role of RopA in secretion and folding of the protease precursor; and 4.) An examination of the role of the Rop loci in virulence in a murine model of streptococcal infection. The proposed studies will utilize recently developed state-of-the-art methodologies for analysis in S. pyogenes and the further characterization of these genes will be important for understanding the function of the protease through understanding when and where it is expressed during infection.